Blood ammonia levels would appear to represent a desirable physiologic set-point for in vitro culture of human tissue. The NH3-N blood levels for normal man range from 5-69 micrograms/dl with a mean of 40.8 plus or minus 10.3 micrograms/dl. In severe hepatic disease in man levels as high as 357 micrograms/dl have been cited. It seems reasonable to suggest that for normal growth, function and differentiation cell culture systems should be poised at NH3-N levels within the physiologic limits of 5-100 micrograms/dl. The NH3-N levels were determined in 65 samples of spent culture media obtained from 12 cultures, as established from a cell line and primary cultures of human prostate tissue. A mean NH3-N level of 1039 micrograms/dl and 2678 micrograms/dl in the prostate and cell line cultures respectively was observed. The major source of ammonia in both FCS and horse serum (HS) supplemented McCoys 5A is derived from the serum component, i.e., 86% FCS 80% with HS. Glutamine and the amino acid mixtures contributed 3-11 and 4-7% of the total NH3-N at the moment of formulation. The NH3-N levels found in cell culture media, as obtained from different laboratories was determined. Abnormally high levels of NH3-N were demonstrated in culture media and/or medium supplements, as derived from 19 different sources encompassing some 56 samples. The overall level of NH3-N in 24 batches of serum obtained from two vendors ranged from 597 to 13,880 micrograms/dl with a mean of 3432.5 micrograms/dl. The daily spontaneous generation of NH3-N in stored tissue culture media, McCoys 5A 10% fetal calf serum - equaled 89 micrograms/dl at 37 degrees, 34.9 micrograms/dl at 24 degrees, 3.7 micrograms/dl at 5 degrees and a minus 2.9 micrograms/dl at minus 20 degrees with -3.4 micrograms/dl being recorded following storage of the media at minus 80 degrees C. Efforts to resolve the obvious "ammonia toxicity" problem via the preparation of low ammonia containing media and its control during actual culture at 37 degrees C are being pursued.